Although the present disclosure will refer to particles throughout much of its specification, the invention includes more generally all classes of small particles including emulsions, macromolecules, viruses, nanoparticles, liposomes, macro-ions and any other solution constituents whose size may lie between a half and a few thousand nanometers. Thus whenever the terms “particle,” “macromolecule,” or “macro-ion” are used, it should be understood they include all of the aforementioned solution-borne objects. The present invention generally involves the characterization of particles and molecules in a liquid sample, and in particular the measurement of fluorescence and light scattering of samples contained within the wells of a multiwell plate. Methods capable of measuring samples directly in these multiwell plates are generally desirable given both the high-throughput nature of the measurements permitting the rapid screening of the individual samples, as well as the reduced sample volume requirements. Standard multiwell plates have 96, 384, or 1536 wells, each well is able to contain a different sample, and all wells, under common operational conditions, may be tested in a single data collection run. In addition, use of these plates obviates the laborious need to clean and dry individual scintillation vials after each measurement. These plates generally have very low volume wells, and commercially available multiwell plate based measurement instruments are capable of measurements from sample volumes of 1 μL or less. These tiny sample volumes are of great benefit when one has a limited amount of sample from which to make measurements, particularly when compared to the 300 μL or larger sized measurement volumes often required by other measurement techniques, such as flow-through fluorescence monitoring and flow-through multiangle light scattering (MALS). Other obvious benefits include the ability to automate the measurement of between 1 and over 1500 samples with little or no human intervention after the sample is prepared and introduced into the plate for analysis. Further labor saving benefits can be achieved, of course, by automated sample preparation robots such as the Freedom EVO® series produced by Tecan (Tecan Trading AG, Switzerland).
Multiwell plates can be used with various optical analysis techniques, most commonly absorbance measurements performed as light is scanned across a plate and the transmitted light is measured by a detector system placed on the opposite side of the plate to the incident light, permitting, thereby a measurement of the absorbance of light by the sample contained in each individual well as described, for example, by A. J. Russell and C. Calvert in U.S. Pat. No. 4,810,096 (Issued Mar. 7, 1989). Measurements of absorbance can enable a calculation of the concentration of the sample contained therein.
Light scattering measurements may also be performed in a multiwell plate as described by Kuebler, et. al., in U.S. Pat. No. 6,519,032 B1 (issued Feb. 11, 2003). In this technique a fine beam of light, generally produced by a laser, is directed to pass vertically through the sample contained in a single well (either from above or below), and scattered light is detected by a detector located beneath the sample well. The apparatus disclosed by Kuebler utilizes a technique generally referred to as dynamic light scattering (DLS) that is also known as quasi-elastic light scattering (QELS) and photon correlation spectroscopy (PCS). When in solution, sample particles are buffeted by the solvent molecules. This leads to a random motion of the particles called Brownian motion. As light scatters from the moving particles this random motion imparts a randomness to the phase of the scattered light, such that when the scattered light from two or more particles is combined a changing intensity of such scattered light due to interference effects will occur. The DLS measurement of the time-dependent fluctuations in the scattered light is achieved by a fast photon counter, generally connected by an optical fiber to collection optics located beneath the multiwell plate. The fluctuations are directly related to the rate of diffusion of the particles through the solvent. The fluctuations are then analyzed to yield diffusion coefficients and, from these, the hydrodynamic radii of the sample.
Another measurement of interest, which is a focus of the present invention, is the detection of fluorescence from particles contained within the liquid sample. In general fluorescence studies involve treating particles, such as chromosomes or proteins with a fluorescence tag or dye. The sample is then irradiated with an excitation light source, the protein tags are excited, causing electrons to rise to a higher energy state, when the electrons from the tagged proteins return to their ground state, they emit photons of a longer wavelength that can be captured by an optical detector, such as a photomultiplier tube (PMT), and analyzed, and thus the tagged proteins can be identified and characterized. Often the spectrum of the fluorescence is measured for even richer information content. Some molecules, most notably proteins, possess intrinsic fluorophores and can be excited with proper light sources. For example, UV light at 280 nm excites both tyrosine and tryptophan residues, while 295 nm light excites only fluorescence from tryptophan residues. Intrinsic protein fluorescence spectrum can be used to assess the tertiary protein structure as the fluorescence peak tends to red-shift as the residues are exposed to more polar environment. The measurement of protein free energy of unfolding can be determined by combining chemical denaturation, generally using urea or guanidine hydrochloride, with measurement of intrinsic fluorescence. The protein free energy of unfolding is an indicator of formulations stability.
Many systems have been developed to measure fluorescence of a sample materials, including directly from animal tissue, lab-on-a-chip technologies, or measuring fluorescence-labeled target molecules immobilized on a solid support. Significantly fewer developments have been concerned with fluorescence studies of liquid samples contained in a multiwell plate, although some patents have explored improvements to the collection of fluorescence data of samples contained therein. For example, U.S. Pat. No. 6,316,774 discloses a fluorimeter where light passes from the source through one of two optical fibers, the selection of which is controlled manually. These fibers permit illumination of the sample contained within a plate or cuvette either through the open top of the chamber or through the transparent closed bottom by reflecting the incoming light from the fiber with a concave, focusing mirror that directs the illumination to the sample chamber. Additional collecting mirrors and optics gather the emission radiation and direct it into optical fibers that carry the light to an optical shutter that permits passage from emitted light gathered either from above or below the sample plate.
Combined illumination and collection optics have been subject of other fluorimetry innovations. U.S. Patent Application No. US 2010/0032582 A1 (published Feb. 11, 2010) by H. Xia, et. al. utilizes a single optical fiber bundle comprising a central illumination fiber surrounded with a plurality of receiving fibers contained within a single bundle, and utilizing a single lens to direct the paths of both the excitation radiation to the sample and receipt of the emission radiation from the sample that is contained within a microfluidic channel. Similarly U.S. Pat. No. 4,678,326 (Issued Jul. 7, 1987) by H. Harjunmaa proposes a bundle of fibers with an emission fiber in the center and utilizing a pair of lenses to collimate excitation radiation through the bottom of cylindrical cuvette in order to avoid illumination of the cuvette walls by the incident light while maximizing the uniformity of illumination of the sample. The same lens configuration collects light emitted from the sample and directs it into a plurality of fibers arranged at specific radial distances surrounding the emission fiber selected so as to reject reflected light from the bottom surface of the cuvette.
In addition to these systems for measuring fluorescence from a multiwell plate, improvements to the optics and light collection and rejection systems have also been considered and are present in the prior art. For example U.S. Pat. No. 7,595,881 by S. W. Leonard, et. al, discloses a useful optical system wherein a shadow disc is placed within the path of emission radiation collected and directed, in free space, by a mirror located below the well plate. By careful positioning and alignment of the optical elements, the shadow disc absorbs light scattered by the meniscus of the sample cell, the remaining radiation is then focused with an aspheric lens onto a detector, improving the overall signal-to-noise of the collected light.
While all of these implementations offer significant improvements over fundamental fluorimeter systems, it is not until the present invention that a simplified system that maximizes the signal to noise ratio while minimizing stray light and permitting the high-throughput analysis enabled by multiwell plates has been possible.